2.
Stephen C. Piper
Abstract
Ionic Fluctuation in Nuttallina californica
Sh w
ABSTRACT
The concentrations of sodium, potassium, calcium, and mag¬
nesium in the blood of Nuttallina californica were measured
and show the typical molluscan trend.
Over a range of experimental salinities from 50 to 1502
sea water, the blood values of sodium and magnesium were the
same as the same jons in sea water, while blood potassjum
and calcium levels were higher, possibly by active regula¬
tion.
The rate of flux was greater in 50 than in 150% sea water
for all ions; the rate series appears to be KNatcaMg?
In exposed animals, sodium and calcium were held constant.
Potassium was initially lowered, magnesium raised; these
changes may be correlated with an observed lowering of
metabolism on exposure to air.
Stephen C. Piper
Ionic Fluctuation in Nuttallina californica
INTRODUCTION
The blood of marine molluscs, with the exception of the
cephalopods, is similar but not identical to sea water in
inorganic composition. The analyses of various workers are
well summarized by Robertson(1964). In general, the concentra¬
tions of sodium and magnesium are the same in the blood and
in sea water while potassium and calcium are more concentrated
in the blood. Except for blood jon values for Katharina tuni¬
cata (Wood,1815) in conditions of fluctuating salinity(Stickle
and Ahokas, 1974), data are nonexistent for blood jonic compo¬
sition for the whole classes Amphineura and Scaphopoda,
Polyplacophoran molluscs are common members of the inter-
tidal fauna of the Pacific Coast of North America. Nuttallina
californica(Reeve,1847) is a small chiton, seldom more than
5 cm long, that is fairly restricted to the middle intertidal
zone of exposed shores from Puget Sound to the Coronado Is¬
lands. Nuttallina californica is usually found in moderately
exposed rocky crevice habitats. It is subject to large fluc¬
tuations of sea water salinity caused by such factors as rain
run-off, evaporation, and summer fog, and is exposed to air
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Stephen C. Piper
Ionic Fluctuation in Nuttallina californica
about ten to twenty hours a day(Ricketts and Calyjn,1968).
This study is an examination of the concentrations of four
inorganic cations in the hemolymph of Nuttallina californica
over a range of experimental salinities and in conditions of
exposure to air.
MATERIALS AND METHODS
Specimens of Nuttallina californica were collected from
the eastern end of the Great Tidepool at Point Pinos, Monterey
County, California during the month of May, 1974. To minimize
possible intraspecific adaptive differences, the animals were
collected from a small area, all at approximately the same
vertical level,+3.5 to15.0 feet above the mean low-low tide.
The animals varied from 2.5 to 8 grams in weight. Äfter being
brought into the laboratory, they were placed in plastic tubs
in sea water of similar salinity, 33.97%, to that sampled at
Page 3
Stephen C. Piper
Ionic Fluctuation in Nuttallina californica
the collection area and designated 100% sea water. Each tub
contained 4 liters of sea water and not more than 17 animals;
2 tubs of the 8 were used for each experimental condition. The
animals were kept in the 100% sea water without food for at
least 44 hours but not more than 68 hours prior to the experi¬
ment. The experiments were carried out in plastic tanks con¬
taining 4 liters of experimental solution and continuously
aerated with porous stone diffusers. The animals were kept
submerged throughout the experiments with a rigid wire screen.
The tanks were covered with aluminum foil lids to reduce evap¬
oration and consequent salinity change and were set in a water
bath maintained at 13° to 14.5° C.; air temperature varied from
14.5° to 15.5° C. inside the covered tubs during the experiment.
Salinities used in this study were 502, 100% and 1502.
To prepare 150% sea water, Instant Ocean, supplied by Aquarium
Systems, Inc., Eastlake, Ohio, was added in sufficient quan¬
tity to bring 100% sea water to 150%. At time 0, the 1002 sea
water was poured from each tub, leaving the chitons attached,
and 4 liters of either 502, 1002, or 150% sea water were added.
In addition, other tubs were drained leaving the chitons exposed
Page 4
Stephen C. Piper
Lonic Fluctuation in Nuttallina californica
to air throughout the experiment.
Measurements of jon levels were made on samples of blood
withdrawn from the area of the heart and pericardial cavity.
The heart, contained within the pericardium, lies directly
beneath shell valves 7 and 8, and the fluid was withdrawn
directly by puncturing the dorsal body wall between these two
valves and inserting a piece of capillary tubing. No signi¬
ficant difference was found between samples taken as indicated
and from positions more anterior. Usually three and occasionally
o animals were used at a sample time. At the same times, sam¬
ples of the sea water solutions were taken. Sampling was for
intervals up to 55 hours.
All samples were analyzed shortly after collection using
a Perkin-Elmer 303 Atomic Absorption Spectrophotometer. The
analytic procedure was based on suggestions given in the Perkin¬
Elmer 303 Analytic Methods Manual. For Kt analysis, aliquots
of 50 or 100 ul. of untreated blood were diluted to 5.05 or
10.10 ml. respectively with a solution of 1000 ppm Na' to re¬
duce effects of jonization. For Ca2t, Mgér, and Naf analysis,
Dliquots of 100 pl. were diluted to a volume of 50.10 ml. with
a solution of 1000 ppm kt to reduce jonization effects. The
Page 5
Stephen C. Piper
Page 6
Ionic Fluctuation in Nuttallina californica
potassium standards were prepared by adding Nat solution, HNO2,
and distilled water to 1000 ppm standard stock solution supplied
by Harleco, Philadelphia, Pa., resulting in standards with 1000
ppm Naf, 12 HNO3; Caef, Mg2t, and sodium standards were made by
adding Kr solution, HNOg and distilled water to 1000 ppm stock
solution, resulting in standards with 1000 ppm K* and 12 HNO..
After 24 hours, the 507and 150% solutions of one set of animals
were replaced with 100% sea water. At the same time, 100% sea
water was added to one set of animals that had been exposed to
Dir.
Although animals were weighed at the time blood was taken, no
significant correlation between animal size and jonic composition
Was observed. A total of 91 Nuttallina californica were examined
during the experiment. Blood was taken from each animal at only
one time.
RESULTS
For chitons maintained in 1002 sea water, the blood con¬
Stephen C. Piper
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Ionic Fluctuation in Nuttallina californica
centrations of Na" and Ngé were not significantly different
from the concentrations of these jons in sea water. Much of
the variability in the data is thought to have been contributed
by "machine drift", during spectrophotometer operation, which
was not completely corrected for by adjusting all the experimen¬
tal blood ion values to values which correspond to sea water
ion values corrected to their mean. The high blood Nat and Mg?t
values for 100% sea water at hour 27.5 probably result from
uncorrected drift. Blood K and Ca2* concentrations were sig¬
nificantly higher than the concentrations of these jons in sea
water (see Table 1). The results of tests for significant dif¬
ferences between blood and 100% sea water jon levels, based
upon the "Student's't-test, are presented in Table 1; changes
in ionic composition of the blood of experimental animals
during the period of observation are shown in Figure 1.
When the chitons were placed in 50%and 150% sea water, the
concentrations of blood Nat and Ng after a period of time
were not different from the concentrations of these jons in
the experimental solutions. K* and Ca2 were higher in the
blood than in sea water at all salinities to at least the 952
Stephen C. Piper
Page 8
Ionic Fluctuation in Nuttallina californica
level of "t test" significance, except the blood Ca2t of animals
in 1003 sea water which was higher with only 90% significance.
The blood and sea water ion levels in 50Zand 150% sea water
were measured outside the range of jon standards and are not
accurate absolute values (eg., note in Fig. I that the measured
Caër mean for 100% sea water is not twice the measured Ca2
mean for 50% sea water). Valid comparisons of relative jon con¬
centrations can be made only at one salinity for a single jon;
however, comparisons of the time required for new equilibrium
Values to be reached are possible, among salinities and among
jons. Due to the variability in the data, highly accurate
equilibration periods or even half-equilibration periods can
not be determined. Rough estimates of the equilibration perjods
were made by graphically determining the time required for the
blood jons to attain their estimated new equilibrium levels.
These estimates are presented in Table 2. The equilibration
periods for all the cations measured were slightly longer in
the 150% sea water than in 50% sea water. In both 50gand 1502
sea waters, Mg- had the longest equilibration period; &at had
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Stephen C. Piper
Lonic Fluctuation in Nuttallina californica
the shortest while Ca2t and Na' had equilibration periods of
intermediate length.
The chitons exposed to air showed no significant differences
from the animals in 100% sea water with respect to their Na'
and Ca2t levels.Blood Mg“, however, was higher in exposed
animals than in animals immersed for the first 16 hours
Blood K, on the other hand,
("Student's" t-test; pX.05).
was lower in exposed animals than in those in 100% sea water to
and then
hour 16 (p.05)
gradually became significantly higher.
The animals kept immersed in 1002 sea water remained in
good condition until hour 39. After this time they were only
slightly attached to the bottom of the tubs, although they
still responded when their feet were touched. Animals exposed
to air remained in good condition during exposure and upon re¬
turn to 100% sea water. Some of the animals in 150% sea water
showed no adverse effects from immersion in water of this
salinity. Others showed poor attachment, but upon return to
100% sea water, these animals all responded when their feet
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Stephen C. Piper
Ionic Fluctuation in Nuttallina californica
were touched. The animals maintained in 50% sea water were in
good condition unt il the thirty-sixth hour and did not
revive upon return to 100% sea water; however, they showed
a foot response throughout the experiment.
DISCUSSION
In this study mean values of ion concentrations in Nut¬
tallina californica in 1004 sea water exhibiteathe typical
molluscan pattern, in that sodium and magnesium concentrations
were almost the same in the blood and sea water, while calcium
and potassium were maintained at higher levels in the blood
than in the ambient sea water.
In addition, in 50% and 150% sea water, the blood jons
eventually assumed the same relationship with the sea water
jons as was observed in 1004 sea water; k' and Caér were main-
tained higher than ambient sea water ion levels at all salin¬
ities, while Nat and Mg’t eventually conformed at all salinities.
A Donnan equi librium situation can not be invoked in explana¬
Stephen C. Piper
lonic Fluctuation in Nuttallina californica
tion of these results since all the diffusible blood cations
would be expected to show raised levels. Selective protein
binding of K" and Ca could explain their elevated levels at
all salinities but Prosser(1973) has concluded that "in all
marine animals the differences remaining after dialyses are
so small compared with the jonic differences between normal
blood and sea water that protein binding must be considered
insignificant as a regulating mechanism." Therefore it appears
that Nuttallina californica actively maintains its blood K-
and Ca“ levels above environmental concentrations at all
experimental salinities.
As indicated by the estimated equilibration periods, the
relative rates of change of all blood ion levels were slightly
smaller in animals in 150% sea water than in 50% sea water. It is
possible that this difference is due to cell damage in the
dilute salinity; Winterstein(1916) has shown that cell injury
leads to increased cell permeability to salts and water. It is
also possible that the difference between the ion changes in
507and 150% sea water is related to the difference in calcium
Page 11
Stephen C. Piper
Page 12
Ionic Fluctuation in Nuttallina californica
concentration in these two experimental solutions. Alexander
et al. (1939) suggested that calcium binding to cephalin mole¬
cules makes a tight molecular structure and thus reduces tissue
permeability, and Martin(1953) pointed out that calcium binding
regulates the degree of coiling of chondroitin-sulfuric acid.
thus altering the pore size of a structure. Therefore, the
greater concentration of calcium in 150% sea water may have
reduced tissue permeability to both water and jons. Another pos¬
sible explanation for the apparent faster rates of jon level
change in 50% sea water may be differences in the ability of
Nuttallina californica to clamp down in the two salinities;
Stickle and Ahokas (1974) have found faster hemolymph osmolality
fluctuations in unattached than attached specimens of Katharina
tunicata. Nuttallina californica was observed to swell con-
siderably in 50% sea water, which may have decreased its ability
to tightly clamp its girdle to the substrate and decrease its
exposure to water of low salinity.
The estimated equilibration periods suggest that the rela¬
tive rates of change of the blood ions under consideration
are : Ktca-NaNgt. In contrast, Stickle and Ahokas(1974)
Stephen C. Piper
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Ionic Fluctuation in Nuttallina californica
found the rate series Mgåtkt Nab ca2t for Katharina tunicata
and Nat Mgetkt Ca for Thais lamellosa(Gmelin,1792), while
Tucker (1970) found Ca2tk Nab Nge for Scutus breviculus(Blain-
ville,1817). The ion flux rate series expected if the concen¬
tration changes were due to integument permeability alone is;
K5 Na CactMg due to the increasing diameter of the hydrated
ions. The similarity of this latter series with that observed for
Nuttallina californica suggests that integument permeability
may be the major factor determining relative rates of change,
although the rate series is based on only 3 points in twelve
hours.
The animals maintained in humid drained tubs for 55 hours
probably did not lose significant amounts of body water during
this interval. The concentrations of Na' and ca2 jons were
never significantly different from the levels of those in
control animals in 100% sea water. The changes in blood K
and Mge levels of animals in exposed conditions are inter¬
esting. K' initially became lower than the same ion of control
animals in 100% sea water, returned to the control level by
Stephen C. Piper
Ionic Fluctuation in Nuttallina californica
the 24°" hour, and continued to rise until it became signif¬
icantly higher than the K' level of immersed animals. Ma?t,
on the other hand, initially became significantly more con¬
centrated in the blood of exposed animals, returned to the
control level by the 2oth hour, and then dropped slightly be¬
low the Mgér level of the control animals. Later it returned
to the level in immersed animals. It has been demonstrated
that moderate increases in potassium jons (507to 1002 and
more) have a stimulatory action on the neuromuscular system
(eg., Wells,1928; Ross and Pantin,1940). Bethe(1927) found that
an increase in potassium in the external medium augmented
Thythmical movements in medusae, phoronid worms and various
crustaceans. Robertson (1953) suggests that the higher muscular
activity of members of the Cephalopoda compared with the Lamel¬
libranchia or Gastropoda may be related to the high k con-
centrations maintained in the blood by members of this class.
In addition, Robertson (1953) points out that solutions of
magnesjum chloride can be used to narcotize marine animals
and has shown that activity and blood magnesjum level are
Page 14
Stephen C. Piper
Page 15
Ionic Fluctuation in Nuttallina californica
inversely related in a series of crustaceans; he suggests that
an increase in blood magnesium and decreased activity are
causally related. A study of the isolated walking legs of
Larcinus indicates that perfusion with a fluid containing 1.5
to 2 times the blood concentration of magnesjum depresses
neuromus cular transmission (Katz,1936) and perfusing with a
fluid containing only 57to 202 of the blood Ng2t concentration
enhances the submaximal muscular response (Boardman and collier.
1946). Findings that the mechanical response to nerve stimu¬
lation varies inversely with the magnesium concentration in
the perfusing fluid have also been reported in three other
decapods, Maia, Panulirus, and Cambarus (Waterman,1941).
The foregoing findings suggest the possibility that the
initial rise in blood magnesium levels and decrease in blood
potassium levels observed when specimens of Nuttallina cali
fornica were subjected to exposed conditions may have parti¬
cular significance to the biology of these animals. Nishi(1974)
has observed low activity in N. californica to be correlated
with low tides, or times of exposure, and high activity to be
Stephen C. Piper
Ionic Fluctuation in Nuttallina californica
correlated with high tides and immersion. The decrease in
blood K“' and increase in Ngé* levels observed when animals
were exposed may act to depress neuromuscular activity and
reduce muscle tone in N. californica during low tides, its
periods of relative inactivity. Robbins (1974) has found the
metabolic rate of N. californica in exposed conditions, as
measured by oxygen consumption, to be approximately 702 of the
rate observed when animals were submerged in sea water. Upon
return to water, the exposed animals did not show the tempo¬
rary increase in rate that would suggest the repayment of an
oxygen debt. Therefore, his results suggest that glycolysis
is depressed during periods of exposure. The changes in k'
and Mg“ levels may be correlated with these suggested modi¬
fications in metabolism. The rise in blood K* level and de¬
crease in blood Mgér level at about the 20th hour (maximum
length of exposure per day) may indicate a physiological
anticipation of a high tide and a period of high activity
by the animals.
lon changes in Nuttallina californica when exposed to the
Page 16
Stephen C. Piper
Ionic Fluctuation in Nuttallina californica
air, a condition with which it is regularly confronted, is
a topic which merits further study.
SUMMARY
1. The concentrations of sodium, potassium, calcium and mag¬
nesium in the blood of Nuttallina californica have been
measured and they show the typical molluscan trend.
Over a range of experimental salinities from 507to 1502
sea water, blood values of Na and Mg were the same as
the respective sea water ion values, while blood potassjum
and calcium levels were higher than the levels of the
same ions in the experimental solutions. Potassjum and
calcium may be actively regulated whereas Na' and Mg
are in passive equilibrium.
3. The rate of flux was apparently greater in 507than in 150%
sea water for all jons; the rate series appears to be:
KtNat-ca2tNg2t.
Page 17
Stephen C. Piper.
Page 18
Ionic Fluctuation in Nuttallina californica
4. In animals in exposed conditions, sodium and calcium levels
were held fairly constant. Potassium was initially lowered,
magnesium raised; these changes may be correlated with an
observed lowering of metabolism on exposure to air.
ACKNOMLEDGMENTS
I would like to express my sincere thanks to the faculty
and staff of Hopkins Marine Station, especially to Dr. John
Phillips for his advice during this study and his assistance
in preparing this paper and to Dr. Robin Burnett for his help¬
ful criticism of this paper. My thanks also go to Dr. John
Martin and Pat Elliott of Moss Landing Marine Laboratory for
their suggestions involving use of the spectrophotometer.
Stephen C. Piper
Page 19
Ionic Fluctuation in Nuttallina californica
LITERATURE CITED
Alexander, A. E., T. Teorell, and C. G. Aborg
1939. A study of films at the lipid/liquid interface. III.
A specific effect of calcium on cephalin monolayers.
Trans. Faraday Soc. 35:1200-1205
Bethe, A.
1927. Der Einfluss der Ionen des Seewassers auf rhythmische
Bewegungen von Meerestieren. Pflüg. Arch. ges. Physjol.
217:456-68
Boardman, D.L., and Henry Oswald Jackson Colljer
1946. The effect of magnesium deficiency on neuromuscular
transmission in the shore crab, Carcinus maenas.
J. Physiol. 104:377-83
Katz, Bernard
1936. Neuromuscular transmission in crabs. J. Physjol.
87:199-221
Martin, M.B.
hes
1953. Chondroitin-sulfuric acid-a linear polyelectrolyte.
Archs. Biochem. 42:181-193
B
Stephen C. Piper
Page 20
Ionic Fluctuation in Nuttallina californica
Nishi, Rae
The diet and feeding habits of Nuttallina californica
1974.
(Reeve, 1847) from two contrasting habitats in central
California with notes on their natural histories. The Veliger
Prosser, Clifford Ladd
1973. Inorganic Ions. Pages 79-110 in C.L. Prosser, ed.
Comparative animal physiology.
Phila¬
delphia, Pa. (W.B. Saunders Co.)
Ricketts, Edward F. and Jack Calvin
Revised
1968. Between Pacific tides. 4th ed.
by JoelW. Hedgpeth.
xiv + 614 pp.; illus. Stanford, Calif. (Stanford Univ. Press)
Robbins, Bruce A.
1974. Aerial and aquatic respiration in the chitons Nuttallina
californica and Tonicella lineata. The Veliger
Robertson, James David
Further studies on
1953.) lonic regulation in marine invertebrates. Journ. exp.
Biol. 30:277-296; 1 fig.
(19 January 1953)
1964. Osmotic and jonic regulation. Pages 283-311 in KarM.
Wilbur and C. M. Yonge,eds. Physiology of Mllusca. vol. I.
New York, N. Y. (Academic Press)
Stephen C. Piper
Page 21

Ionic Fluctuation in Nuttallina californica
Ross, D. M., and Carl Frederick Abel Pantin
1940. Factors influencing facilitation in Actinozoa. The
action of certain jons.Journ. exp. Bjol. 1/261-73 ; 6 figs. (25 July 1939)
Stickle, William B., and Robert Ahokas
1975 Tidal salinity effects of mollusc hemolymph compo¬
sition. Comp. Biochem. Physiol. 50: 291-296.
Tucker, Lois E.
1970. Effects of external salinity on Scutus breviculus
"Gastropoda, Prosobranchia)--I. Body weight and blood com¬
position. Comp. Biochem. Physjol.36:301-319; 9 figs. (16 February 1970)
Waterman, Talbot Howe
1941. A comparative study of the effects of jons on whole
nerve and isolated single nerve fiber preparatjons of
crustacean neuromuscular systems. Journ. cell. comp. Physjol.
18109-26 ; 8 figs.
Wells, George Philip
1928. The action of potassium on muscle preparations from
invertebrates. Brit.Journ.exp. Bjol. 5:258-82 ; 12 figs. (8 December 1927)
Winterstein, H.
1916. Beiträge zur Kenntnis der Narkose--IV. Narkose
und Permeabilität.
Biochem. Z. 75:71-100
Stephen C. Piper
Page 22
lonic Fluctuation in Nuttallina californica
Figure Caption
Fig. 1. Changes in ionic composition of the blood of ex¬
perimental animals during observation.
— Blood ionic levels in animals in 100% sea water

o Blood
onic levels in animals in 50% sea water
— Blood ionic levels in animals in 150% sea water
———— Blood jonic levels in animals in exposed conditions
Vertical lines represent standard devjations
Stephen C. Piper
Ionic Fluctuation in Nuttallina californica
TABLE CAPTIONS
lable 1. Concentration of jons in blood of Nuttallina
californica compared to ion concentrations in 100% sea
water in which the animals were maintained. The "Student's'
t-test was used to determine significance levels. All
ion values are given as ma/liter.
lable 2. Estimated equilibration periods of ions in 50Zand
150% sea water. Equilibration periods were estimated by
determining the time required for the blood jons to attain
their estimated new equilibration levels.
Page 23
Stephen C. Piper
Ionic Fluctuation in Nuttallina californica
Table 1
Ca?t
Mg
Nat
Kt
Blood concentration
10243.8 1225.4 482.0 499.6
Sea water concentration
10234.4
1220.6 434.4 456.4
Number of blood samples
16
16
13
16
Number of water samples
6
Blood sample standard
16438.0
145.7
131.9 +46.4
deviation
Water sample standard
1754.8
162.0 141.4 +40.9
deviation
Significance level
N.S.
N.S.
99.9% 90.0%
Stephen C. Piper
Ionic Fluctuation in Nuttallina californica
Table 2
Na?
Kt
Na ca?t
Equilibration Time in hour
50% sea water
3-5 3-5 3-7 8-12
4-8 6-10 6-10 10-14
150% sea water
Geve Heer
lenie Fluctuatioo  M.calfcnica
700
oo

50/
600
900
waer

800
1500

1507 sea
1007.

———
wotec
sea watee
700
100

300
600
50' sea water
St
50
1507
1007sea
sea
waer
400
00
water



1800
300
50 sea wate
200
170

15
1507.
14
—sa


water
13
130
12

07 sea

wat
200
1ook sea
ff
water
10
800
osea
—° 50¼
P
water
sea wäte




6o0l
1 20 30 0 50 60
20 30 40 50 60
Time i Hrs.
Time jo Hes.
e